Anti-Inflammatory Activity and Structure-Activity Relationships of Brominated Indoles from a Marine Mollusc

Abstract

Marine molluscs are rich in biologically active natural products that provide new potential sources of anti-inflammatory agents. Here we used bioassay guided fractionation of extracts from the muricid Dicathais orbita to identify brominated indoles with anti-inflammatory activity, based on the inhibition of nitric oxide (NO) and tumour necrosis factor α (TNFα) in lipopolysaccharide (LPS) stimulated RAW264.7 macrophages and prostaglandin E2 (PGE2) in calcium ionophore-stimulated 3T3 ccl-92 fibroblasts. Muricid brominated indoles were then compared to a range of synthetic indoles to determine structure-activity relationships. Both hypobranchial gland and egg extracts inhibited the production of NO significantly with IC₅₀ of 30.8 and 40 μg/mL, respectively. The hypobranchial gland extract also inhibited the production of TNFα and PGE2 with IC₅₀ of 43.03 µg/mL and 34.24 µg/mL, respectively. The purified mono-brominated indole and isatin compounds showed significant inhibitory activity against NO, TNFα, and PGE2, and were more active than dimer indoles and non-brominated isatin. The position of the bromine atom on the isatin benzene ring significantly affected the activity, with 5Br > 6Br > 7Br. The mode of action for the active hypobranchial gland extract, 6-bromoindole, and 6-bromoisatin was further tested by the assessment of the translocation of nuclear factor kappa B (NFκB) in LPS-stimulated RAW264.7 mouse macrophage. The extract (40 µg/mL) significantly inhibited the translocation of NFκB in the LPS-stimulated RAW264.7 macrophages by 48.2%, whereas 40 µg/mL of 6-bromoindole and 6-bromoistain caused a 60.7% and 63.7% reduction in NFκB, respectively. These results identify simple brominated indoles as useful anti-inflammatory drug leads and support the development of extracts from the Australian muricid D. orbita, as a new potential natural remedy for the treatment of inflammation.

Keywords: Muricidae; NO inhibition; Tyrian purple; inflammation; isatin; marine natural products.

Figure 1

Liquid chromatography mass spectrometry (LC-MS) chromatographs of several extracts from Dicathais orbita showing brominated indoles identified by mass spectrometry (molecular ions for Br⁷⁹, Br⁸¹): (a) chloroform extract of the hypobranchial gland; (b) chloroform extract of the egg mass; (c) degraded chloroform extract of the hypobranchial gland; and (d) methanol extract of the hypobranchial gland. Mass spectra for the main brominated indoles are shown in Supplementary Figure S1.

Figure 2

Gas chromatography mass spectrometry (GC-MS) of the chloroform extracts from Dicathais orbita showing brominated indoles identified by mass spectrometry (molecular ions for Br⁷⁹, Br⁸¹). GC-MS chromatograms of the (a) hypobranchial gland chloroform extract; (b) purified fraction containing tyrindoleninone, with the associated mass spectral fragmentation pattern. The brominated compounds were identified based on characteristic mass isotopic patterns for brominated indoles (Br⁷⁹ and Br⁸¹), and matched to the NIST02 database. The structures for the main compounds tested are provided in Table 2. The mass spectra for the brominated indoles in the extract are shown in Supplementary Figure S2.

Figure 3

NO inhibition in RAW264.7 mouse macrophages stimulated with lipopolysaccharide (LPS) and treated with extracts from Dicathais orbita, purified brominated indoles, and synthetic analogues. Data are expressed as % inhibition of NO production relative to the negative control DMSO: (A) extracts from the hypobranchial glands (HBG) and egg capsules; (B) the purified natural products tyrindoleninone and tyriverdin; (C) the monomer synthetic indoles isatin, 5-bromoisatin, 7-bromoisatin, and 6-bromoindole; (D) the dimer synthetic indoles indirubin, 6-bromoindirubin, and 6,6 dibromoindigo. Data shown are means ± SEM from three separate experiments performed in triplicate. The symbols above the bars indicate statistically significant differences in the amount of nitrite in the treatments compared to the DMSO control. * p < 0.05, # p < 0.01, ^ p < 0.001, + p < 0.0001 versus untreated, stimulated cells (LPS + DMSO).

Figure 3

NO inhibition in RAW264.7 mouse macrophages stimulated with lipopolysaccharide (LPS) and treated with extracts from Dicathais orbita, purified brominated indoles, and synthetic analogues. Data are expressed as % inhibition of NO production relative to the negative control DMSO: (A) extracts from the hypobranchial glands (HBG) and egg capsules; (B) the purified natural products tyrindoleninone and tyriverdin; (C) the monomer synthetic indoles isatin, 5-bromoisatin, 7-bromoisatin, and 6-bromoindole; (D) the dimer synthetic indoles indirubin, 6-bromoindirubin, and 6,6 dibromoindigo. Data shown are means ± SEM from three separate experiments performed in triplicate. The symbols above the bars indicate statistically significant differences in the amount of nitrite in the treatments compared to the DMSO control. * p < 0.05, # p < 0.01, ^ p < 0.001, + p < 0.0001 versus untreated, stimulated cells (LPS + DMSO).

Figure 4

Percent inhibition of TNFα in LPS stimulated RAW264.7 macrophages after treatment with Dicathais orbita hypobranchial gland (HBG) extract and associated brominated indoles: (A) chloroform extract and tyrindoleninone, purified from the HBG extract; (B) synthetic isatin and indole compounds. Data shown are means ± SEM from three separate experiments performed in triplicate. The symbols above the bars indicate statistical significance of the differences in the amount of TNFα in the samples compared to the DMSO control. * p < 0.05, # p < 0.01, ^ p < 0.001, + p < 0.0001 versus LPS + DMSO.

Figure 5

PGE2 inhibition in calcium ionophore stimulated 3T3 ccl-92 fibroblasts after exposure to a chloroform extract from the hypobranchial glands of Dicathais orbita and the associated brominated compounds 6-bromoisatin and 6-bromoindole. Data shown are means ± SEM from three separate experiments performed in triplicate. Symbols above the bars indicate statistically significant differences in the amount of PGE2 produced in the samples compared to the DMSO treatment. * p < 0.05, # p < 0.01, ^ p < 0.001, + p < 0.0001 versus LPS + DMSO.

Figure 6

The inhibition of nuclear factor kappa B (NFκB) translocation: (A) representative images of the RAW264.7 cells obtained by an Olympus FV i10 confocal microscope showing the effect of each treatment on the translocation of the p65 subunit of the NFκB stained with Alexa fluor 594 (red fluorescence) into the nucleus. The inhibitor subunit was stained with Alexa fluor 488 (green fluorescence) to highlight the inactivated NFκB in the cytoplasm. 4’,6-Diamidino-2-phenylindole (DAPI; blue) was used to stain the nucleus. Scale bar set to 10 μm; (B) LPS-induced activation of NFκB in RAW264.7 showing the average intensity of the NFκB fluorescence (red) inside the nucleus; (C) mean % NFκB inhibitory activity of the hypobranchial gland (HBG) extract from Dicathais orbita and the synthetic compounds 6-bromoisatin, isatin, and 6-bromoindole, based on the reduction of fluorescence intensity relative to the DMSO + LPS stimulated control. All test compounds/extracts were tested at a final concentration of 40 μg/mL. Data shown are mean ± SEM from three separate experiments. “+” = p < 0.0001 versus LPS + DMSO. All data were obtained using the image processing and analysis software ImageJ (https://imagej.nih.gov/ij/).